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Genetic Engineering Publications - GEG Tech top picks
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The authors establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. This strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches. 
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In this work, the authors demonstrate that nuclease-inactive S. pyogenesCRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. They show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation ofACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. They also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Their results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.